The Leica EM AFS discloses a device according to the existing art. A Dewar vessel is filled with liquid nitrogen, the Dewar neck having a chamber that is cooled to a specific temperature. The desired temperature is set via a control circuit and built-in heating elements. The substitution process usually begins at approximately −90° C. The frozen specimen is transferred into the chamber, for which purpose several different containers can be provided with which the specimens are immersed into a substitution medium, usually acetone or methanol. At this low temperature the slow process of substitution begins, in which the frozen water in the specimen is replaced by the solvent without the occurrence of recrystallization. During this process the temperature is then slowly raised, and the medium is exchanged and ultimately replaced with a low-temperature embedding medium. A UV lamp is placed onto the chamber for polymerization of the low-temperature embedding medium. The various containers for cryosubstitution and embedding are disclosed in the catalog for the Leica EM AFS. All the containers disclosed therein have the disadvantage that a loss of specimens can occur during the process of cryosubstitution and/or embedding.
There are a variety of methods and devices for effective freezing of specimens. The high-pressure freezing method yields the best results at present. After initial freezing, the frozen specimens are located in specimen carriers. These specimen carriers are illustrated on page 6 of the Leica EM PACT catalog. Detaching the small specimens (usually 0.2 mm thick and 1.2 mm in diameter) from the mount, manipulating these specimens in the substitution medium, and transferring them into an embedding mold are critical steps that often result in loss of a sample.
U.S. Pat. No. 6,516,620 discloses an apparatus for high-pressure freezing of specimens. Here the specimen carrier is clamped in on all sides into a holder, and is impinged upon by the corresponding pressure from one side. A cooling medium is sprayed from the outside onto all sides of the specimen holder in order to achieve the requisite low temperature.
U.S. Pat. No. 6,536,219 discloses an apparatus and a method for precise cryosubstitution of tissue samples. The apparatus possesses multiple depressions of different sizes and shapes for embedding. With this apparatus as well, however, the possibility that a loss of specimens may occur cannot be ruled out.